574 | Nature | Vol 577 | 23 January 2020
Article
(Fig. 3 and Extended Data Table 1). The complex reveals a molecule of
cA 4 bound at the dimer interface. Comparison of the cA 4 -bound and apo
structures reveals a substantial movement of a loop (comprising resi-
dues 82–94) and subsequent α-helix to bury cA 4 within the dimer. These
loops adopt variable or unstructured conformations in the various apo
protein structures. Once bound, the ligand is completely enclosed by
the protein—a considerable accomplishment when one considers the
relative sizes of protein and ligand (Fig. 3b). Superimposition of the
cA 4 ligand on the apo-protein structure reveals that the binding site is
largely preformed, with the exception of the mobile loops that form
the lid (Fig. 3c). The overall change is like two cupped hands catching
a ball, with the loops (fingers) subsequently closing around it.
The cA 4 molecule makes symmetrical interactions with each mono-
mer of AcrIII-1 (Extended Data Fig. 5). Arginine R85 on the loop from
one monomer interacts with the distant half of the cA 4 molecule and
appears to ‘lock’ the closed dimer. Other important interactions are
made with main-chain L92, I69 and N8, and side-chain R66, N8, Q81, S11,
T50, S49 and N13, most of which are semi or fully conserved (Extended
Data Figs. 1, 5), suggesting that they have important roles in cA 4 binding
and/or catalysis in this whole family of enzymes. At two positions, on
opposite sides of the ring, the 2′-hydroxyl of the ribose is positioned
correctly for in-line attack on the phosphodiester bond, consistent
with the observed bilateral cleavage (Fig. 3d). The catalytic power of
the AcrIII-1 family probably derives from active-site residues that posi-
tion the 2′-hydroxyl group for in-line nucleophilic attack, stabilize
the transition state and protonate the oxyanion leaving group^24. For
the AcrIII-1 family, the absolutely conserved residue H47 is suitably
positioned to act as a general acid and fulfil the latter role (Fig. 3d). To
test this hypothesis, we assayed variant H47A of AcrIII-1. The variant
enzyme suffered a more than 2,500-fold decrease in catalytic power,
which could be partially reversed by chemical rescue with 500 mM imi-
dazole in the reaction buffer (Extended Data Fig. 6). We also noted that
the conserved residue E88, situated on the tip of the loop that covers
the binding site, is positioned close to the H47 residue of the opposite
subunit. When mutated to alanine, the catalytic rate was reduced by
84-fold to 0.064 min−1 (Extended Data Fig. 6b), consistent with a role
for E88 in positioning H47 and/or increasing the pKa of the catalytic
histidine residue to enhance catalysis^25.
By targeting a key signalling molecule, a single AcrIII-1 enzyme should
have broad utility in the inhibition of endogenous cA 4 -specific type
III CRISPR systems in any species. Of the CRISPR ancillary nucleases
studied to date, most are activated by cA 4 ; activation by cA 6 appears
to be limited to certain bacterial phyla, including the Firmicutes and
Actinobacteria^21. Recently, a type III Acr (AcrIIIB1) has been reported
that appears to function by binding and inhibiting the type III-B effector
complex^26. Two other Acr proteins with enzymatic functions have been
described: AcrVA1, which catalyses CRISPR RNA (crRNA)-mediated
cleavage of Cas12a^27 , and AcrVA5, which acetylates the site in Cas12a
that senses the protospacer-adjacent motif (PAM) of target DNA^28.
These and other Acrs target a protein (or protein/nucleic acid complex),
implying a requirement for specific interactions that could be evaded
by sequence variation. This is not a limitation of AcrIII-1.Phylogenetic analysis of AcrIII-1
The gene encoding AcrIII-1 is found in representatives of at least five
distinct viral families, making it one of the most widely conserved of all
archaeal virus proteins^29 (Extended Data Fig. 1 and Supplementary Data 1).
The distribution of AcrIII-1 in archaea is sporadic but covers most of
the main lineages (Supplementary Data 1), and is typically adjacent to
open reading frames (ORFs) from mobile genetic elements rather than
CRISPR loci. A good example is the STIV integrated into S. acidocal-
darius genomes^30. AcrIII-1 is also present in several bacteriophages of
the order Caudovirales, and there are many instances of acrIII- 1 genesacbapod
H47H47′2.8 Å
3.0 Å3.1 Å
168°3.3 Å
126°4.2 Å 98°3.0 Å 170°Fig. 3 | Structure of AcrIII-1 bound to cA 4. a, Superimposition of the apo SIRV1
gp29 structure (salmon) and the same protein in complex with cA 4 (purple),
highlighting the movement of the loop and α-helix upon cA 4 binding. cA 4 is
shown coloured by element. b, Surface representation of the structure of SIRV1
gp29 (purple) in complex with cA 4 , emphasizing the complete burial of the
ligand. c, Surface representation of the apo structure of SIRV1 gp29 (salmon)
with cA 4 in the position observed in the structure of the complex, indicating
that the binding site is preformed. d, Structure of cA 4 bound to SIRV1 gp29. The
two active-site histidine residues (H47A and H47A′, from each monomer of the
dimer; modelled on the basis of the position of the alanine side chain in the
H47A variant crystallized with cA 4 , and coloured to represent residues from
different monomers) are in suitable positions to act as the general acid,
protonating the oxyanion leaving group. The corresponding ribose sugars
have 2′-hydroxyl groups suitably positioned for in-line nucleophilic attack on
the phosphodiester bond. In the cA 4 ligand, carbon atoms are shown in green,
phosphates in orange, oxygens in red and nitrogens in blue.